Mitochondria provide energy, buffer calcium, and sequester cell death-inducing molecules, and mitochondrial dysfunction is implicated in various neuropathologies. Especially in neurons, mitochondria are highly dynamic organelles that constantly move, divide, and fuse. This proposal investigates the regulation of mitochondrial fission and fusion in neurons, which are antagonistic processes carried out by large GTPases similar to dynamin. Mutations in two of these enzymes, Opal and Mfn2, are responsible for hereditary neurological diseases. While a proper balance of mitochondrial fusion and fragmentation is clearly important for neuronal survival, some fragmentation is necessary for axonal and dendritic transport of mitochondria, and consequently for the development and function of synapses. We have found that shape changes of mitochondria are controlled by an opposing protein kinase and phosphatase that are localized to the outer mitochondrial membrane via specific targeting/regulatory subunits. On the phosphatase side, Bp2 is a neuron-specific, postnatally induced protein phosphatase 2A (PP2A) regulatory subunit mutated in spinocerebellar ataxia type 12. The alternatively spliced N terminus of Bp2 mediates translocation of the PP2A holoenzyme to the mitochondrial surface, where PP2A accelerates cell death, apparently by fragmenting mitochondria. The kinase opposing PP2A/Bp2's effect on mitochondrial morphology and survival is cAMP-dependent protein kinase (PKA) anchored to the OMM via A kinase anchoring protein (AKAP)121. Aim 1 investigates the mechanism by which outer-mitochondrial PP2A and PKA control neuronal survival. In Aim 2, we will identify the relevant substrates and phosphorylation sites among mitochondrial fission/fusion enzymes. Aim 3 addresses the role of PP2A/PKA-dependent mitochondrial restructuring in the delivery of mitochondria to and development of dendritic spines. Finally, Aim 4 characterizes PP2A/Bp2 knockout mice in terms of mitochondria and synapse morphology and resistance to ischemic injury. These studies will advance our understanding of how shape transitions of mitochondria are regulated, and how this affects vulnerability of neurons and the establishment of functional connections between them. Our studies may ultimately lead to better therapies for stroke and neurodegenerative disorders.